Creatures from the Black Lagoon: Lessons in the Diversity and Evolution of Eukaryotes

I’ve organized this lecture into several parts, beginning with a history of biological classification of life — especially in the context of microorganisms. I also want to talk about the diversity and lifestyle of microbes (protists in particular) that live without oxygen and why this is relevant to the evolution of life — especially with respect to eukaryotic evolution. Then I’m going to discuss how the majority of microbial life is yet to be discovered, and how one might find new microbes of interest to the study of evolution. And lastly, I’ll talk about some new local monsters we've found — microbial creatures from the Black Lagoon, where they live (just in case you want to check them out), and why they could change our understanding of eukaryotic evolution.

So, to start, I think when I first I learned about evolution there was always an implied "march of progress," or a linear progression from one form of life to a next, higher and higher, up the evolutionary ladder. This notion usually begins with some primordial soup — progressing to bacteria — to protist — to plant to animal — bugs — to us. But we know this is false. Evolution is branching — or at least that’s been the thinking in the past 150 years. I’ll be talking more about this, but in order to do good research in evolution, we need to examine where these evolutionary "stories" come from — what are the assumptions? I’m going to try to address several of these assumptions and misconceptions by showcasing some of the scientists who formed the basis of the classification of microorganisms.

Aristotle
Let's start in ancient Greece with Aristotle — in addition to many other contributions to science, he was the first to articulate a dichotomy of life, classifying all life as being either plants or animals. This ancient world view of biology persists today, and misinforms our perspective in terms of evolution, not to mention the structuring of scientific research (like botany departments and zoology departments). It was a few thousand years later that the microscope was invented, and animal/vegetable/or mineral types of classification were hardly enough to classifiy the new microorganisms, first called "animacules." Microbes are often non-descript rods, or spheres. How then does one classify microbes, particularly when they don't really look different from one another like plants look different from animals? More about how to do that later, but on to more history.
 

		Carolus Linnaeus Linnaeus lived in the 18th Century and created the familiar hierarchical classification scheme of life: kingdom, family, class, order, family, genus and species. Again, this classification scheme is based on how organisms looked — essentially their phenotype. Although this heirarchy within the plant and animal kingdoms was an improvement — an attempt to standardize classification whose legacy persists today — there were still no good working definitions of "kingdoms" or "species" which could apply to microorganisms when they were discovered. Even today the conception of a species of bacteria is hard to define, in the same way an animal species is defined.
		Charles Darwin We are all probably familiar with Charles Darwin, 19th century father of natural selection and evolution, but he also called for a framework or geneology of organisms in order to study evolution. Darwin helped to develop the concept of a geneology of life with branching lineages. But we attribute the conception of evolutionary relationships among organisms with a tree — a geneology — to Ernst Haeckel at roughly the same time.
		Ernst Haeckel Ernst Haeckel also worked in the mid-19th century. Haeckel was the first to coin the term "protist" or "Protista," although his definition also included the bacteria.

Haeckel's Tree of Life Ernst Haeckel is attributed to be the first to describe the evolutionary relationships among living organisms, a geneology of life, as analogous to a tree. Haeckel, interestingly, described all living things as falling into not just two kingdoms (plants and animals), but also a third kingdom for microorgansims — the Protista. There are several other interesting features of this vision — for example, Haeckel postulated a common origin for all life (plants, animals and microbes). This is still a common assumption, but it makes sense with modern molecular evidence as well. OK, let's fast forward a hundred years or so. How do we classify microbes today, especially when they have no particular morphology or shape to speak of. Scientists had all but given up on classifying microbes based on their evolutionary relationships (a phylogeny) until Carl Woese.
The Woesian Revolution Woese based his classification on molecules, not how organisms look or act. This transition from classification based on phenotype (taxonomy) to one based on genotype enabled him to determine the evolutionary relationships (a phylogeny) among bacteria — something other researchers had all but given up on. Woese's work was founded on the principle suggested in 1965 that "molecules are documents of evolutionary history." Basically, DNA can be thought of as molecular fossils. At the University of Illinios in the late 1970s, Woese wanted to determine evolutionary relationships among microorganisms, and in the process, he and colleagues discovered a huge split in the "prokaryotes" — as big a genetic difference as that between prokaryotes and eukaryotes. Woese orignally thought that these were primitive organisms and so called them the Archaea. Later studies showed that the Archaea were actually more related to the eukaryotes in many aspects that to the bacteria. So, rather than five kingdoms of life, Woese argued for three domains (Eucarya, Archaea, and Bacteria). This conception of three domains of life and two domains of prokaryotes, rather than the standard prokaryote/eukaryote dichotomy, really shook up the study of the evolution of life. So how did Woese arise at this remarkable conclusion? First he needed a molecule to study from all organisms — ribosomal RNA (rRNA).
Why use rRNA? Ribosomal RNA (rRNA) is an RNA component of the ribosome, the cellular machine which translates the DNA genetic code to amino acids, and subsequently, proteins. The rRNA genes are ubiquitous in all life, being conserved enough to identify, yet containing enough variabilty to determine evolutionary relationships. But Woese has had a hard time selling this idea, mostly because everyone has been trained to view life as prokaryotes/eukaryotes, plants/animals, or the updated version of that &emdash; the five kingdom view.

Five Kingdoms or Three Domains? In the 1960s, Whittaker added one kingdom (fungi) to Aristotles's view of life (plants and animals) but still relegated all microbes to either the Protista (eukaryotic microbes) or the Monera (bacteria). Something from this figure (and view) should be obvious. Why should we emphasize macroscopic life over microscopic life? In terms of life on Earth, we still live in an age of microbes. For the majority of geologic time, some 3-4 billion years, life has evolved — and at least three billion has been solely microbial.

The five-kingdom view is still taught, as I'm sure many of you know. Carl Woese started a revolution in the classification of life, much like Copernicus did with his idea that the Sun doesn't revolve around the Earth. We, as humans, possess a lot of unique characteristics such as the ability to understand the world we live in. But we are not the pinnacle of evolution — nothing is. Evolution is about change through time, not about ranking life based on some criterion. Besides, since the bulk of genetic diversity of life on Earth is been microbial (note where we are in Woese's tree), shouldn't they get attention simply for that reason? If you take nothing else from my talk, stop thinking about biology in terms of five kingdoms.
		Since Woese's early trees, we've added a lot of sequences as you can see in the figure to the left. I'd like to switch now from talking about microbes in general, to specfically eukaryotic microbes (that is, microbial cells which possess a membrane-bound nucleus and tend to be unicellular). What's the current picture of eukaryotic phylogeny?

The Evolution of the Eukaryotes As in the prokaryotic world, one can see from this schematic tree of the eukaryotes (below), that the majority of eukaryotic life is, in fact, microbial (protists). There is far more phenotypic and genotypic diversity in all the protist groups combined than within the plant, animal or fungal kingdoms. How many kingdoms of eukaryotes are there? This is still largely unknown. If we use the genetic measure, we come up with roughly 70 eukaryotic kingdoms, only some of which are shown here. Further, because of our bias toward macroscopic life, most of the biology of these protists is largely unknown. How did the eukaryotes evolve? To understand that, we need to visualize the early Earth. II. Life in anoxic worlds Early Earth We know from the geologic record that the early Earth was a fireball and the atmosphere was quite different from what it is today — in fact, no oxygen was present. All oxygen was produced biogenically by microorganisms several billion years after the earth was formed. So for roughly half of Earth's history, there was no oxygen atmosphere. When microbes began producing oxygen in such quantities that it began to saturate the atmosphere, it was the biggest environmental catastrophe in Earth's history. Some of the oldest fossils belong to what look like photosynthetic bacteria — bacteria which breathe carbon dioxide and exhale oxygen. So this process happened early in Earth's history. Microbes made the world. Think about it — oxygen is actually quite poisonous, generating hydrogen peroxides and oxygen radicals. Many microbes don't have a way of dealing with poisonous oxygen. So they avoided it . . . and they still do today. Fossil Redbeds This is a rock from a fossil redbed. The red color derives from oxidized iron, like rust. When the percentage of oxygen in the atmosphere became high enough, iron mineral deposits became oxidized (red). Reduced iron compounds like iron sulfides in contemporary anoxic environments look black. How did this happen? Microbes. Specifically, bacteria called cyanobacteria (or blue-green algae) — they use light as energy, breathe carbon dioxide and generate oxygen. Cyanobacteria This is one example of a cyanobacterium, Spirulina. Spirulina is pretty common in many aquatic habitats. If you've ever seen a green scum on the surface of soils/sediments, that is cyanobacteria. Think of how many cells you would need to completely change the composition of the Earth's atmosphere, as happened a few billion years ago. Cyanobacteria are found all over the world. In fact, in Berkeley you can find them in grocery stores as . . . Spirulina Smoothies! Evolution of Oxygen This graph shows the percentage of oxygen in the atmosphere over time and indicates when red beds would have formed. The first appearance of red beds is at two billion years, which is also the oft-cited age of eukaryotes (cells with a membrane-bound nucleus). Why? The oldest eukaryotic fossils are coincidentally about the same age. But do eukaryotes require oxygen? We were all taught about simple cells like bacteria which use glycolysis or fermentation alone to generate ATP (the energy currency of the cell). Then as evolution progressed, eukaryotes developed the ability to harness oxygen via the mitochondrion, the energy powerhouse of the cell. This is another misconception. First, bacteria invented oxidative respiration — eukaryotes just acquired the ability, by acquiring a bacteria cell by endosymbiosis — this later became the mitochondrion. Also, many eukaryotes do live prosperous lives without oxygen (or without mitochondria for that matter). Here's one below (Giardia), that hopefully no one has had a personal relationship with: Giardia is an aquatic protist, and a parasite that major cause of dysentery world wide. Hopefully no one here has encountered it while camping. Giardia has no mitochondrion and it likes to avoid oxygen. Why? Oxygen is an abundant source of energy, but in many aquatic environments, it gets quickly used up by microorganisms. Many bacteria, however, can respire or "breathe" alternatives to oxygen — sulfur, hydrogen, nitrogen for example. These bacteria are called anaerobes (more on this later). Environments lacking oxygen (anoxic) harbor many of these types of bacteria &emdash; billions per teaspoon of sediment or muck. In many cases, the anoxic environments harbor more microorgansisms than oxic ones. Giardia survives by eating anaerobic bacteria in these rich anoxic environments. But where on Earth are these "extreme" anoxic environments? Where would one go to find them? Contemporary anoxic worlds Virtually anywhere! Check it out — the Black sea is the largest land-locked anoxic basin. Can we find more organisms like Giardia which might still live in anoxic worlds, and still be unknown to us? How could we do it? III. Uncultivated Microbial Eukaryotes What is the extent of eukaryotic diversity? How do we find life on Earth? Why should we care? Well, in terms of studying evolution, our knowledge of modern eukaryotes informs our knowledge of their evolution. An incomplete understanding of diversity of today's eukaryotes results in incomplete evolutionary trees. Characteristics of eukaryotes that live without oxygen Most known anaerobic protists are pathogens. One could argue that the only reason we know about the ones we do is becuase they affect us — they cause disease. Anaerobic protists have evolved multiple times (which is termed "polyphyletic") but since the most basal eukaryotes are anaerobes, we believe the original eukaryotic cell evolved in an anoxic world. How might we look for eukaryotes still alive today, which might be more genetically closer to those ancestral types, than to more recently evolved groups like us? Identifying protists by microscopy One way we could do this would be by microscopy. Antoine van Leeuwenhoek, a lay person (not a card-carrying scientist), really began the study of microbiology in the 17th century with the use of one of the first microscopes. van Leewenhoek described the first microorganisms — he called them "animalcules." People still identify microbes based solely on microscopic descriptions today. These descriptions can read like field guides. Though our microscopes have gotten a lot more sophisticated since van Leewenhoek (e.g., electron microscopes), are there other ways to find these relict eukaryotes? Identifying protists by cultivation Well, we could grow them. We know Louis Pasteur for a lot of work with microbiology, such as germ theory and pasteurization. But he was also the first to describe organisms which were able to grow in sealed flasks without "air." This was truly a first, as people at that time believed that all organisms needed air, meaning oxygen, to grow. But we encounter problems when we rely only on visualizing or growing organisms in order to identify them. First and foremost, it can be really hard! Think if we had to grow every animal in the zoo or every plant in the forest in order to know they were there. Plus, we don't do a really good job at growing most microbes. It's estimated that only about 1% of all microbes in the natural world have been grown. Fortunately, there's a better way to identify organisms — not by how they look or how they grow, but by their DNA. Identifying protists by their genotype DNA. We hear a lot about DNA, the genetic material of all organisms is always in the news. You've probably heard about DNA fingerprinting to identify suspects in crimes, or prove paternity. Well, this method of DNA fingerprinting has been used extensively in the past 15 years or so to identify uncultivated bacteria and archaea. And, using these new molecular techniques, we've discovered that the microbial diversity of bacteria and archaea has been grossly underestimated. Our study, and the rest of the work I'll present today is the first to look for uncultivated microbial eukaryotes, or protists, by their DNA fingerprints. How do we do this? A tablespoon of mud We start with about a tablespoon of mud — remember that this can contain several billion microbes. This is mud from Berkeley Aquatic Park. Then we extract the DNA from the mud. By the way, the black color of the mud is a good indicator that there is no oxygen at that layer. Reduced iron complexed with sulfur produces something called pyrite — a dark black precipitate. Xeroxing DNA The polymerase chain reaction, or PCR, is basically a way to xerox DNA so that we have enough to work with in the lab. Many microbes are rare and we need to amplify their DNA signals to be able to find them. In our work we're xeroxing a specific gene — the gene for rRNA. Remember this is what Carl Woese used to relate different microbes to each other and construct a tree of life. Patterns in DNA Once we amplify rRNA genes from a given environmental sample, we need to sort them by type. One tablespoon of mud could have several billion microbes, perhaps representing 1,000 different types! To categorize these different types, we use a technique called RFLP (restriction fragment length polymorphism) which generates unique patterns or fingerprints corresponding to unique organisms. At right is an example showing the DNA fingerprints of the different types of sequences (organisms) we find. Notice the different banding patterns. Aligning genes Once we get the DNA sequence, we line up the sequences and compare them to known organisms. The rows correspond to an rRNA sequence from a particular organism; the column relates the exact nucleotide (A,T,C,G) at a specific position in the sequence of the gene. Note the conserved/variable regions. Constructing a phylogenetic tree Then, from this alignment of DNA rRNA sequences, we use computer models to simulate the evolution of sequences. We infer that the evolution of the sequences corresponds to the evolution of the organisms. In this way, we are able to determine evolutionary relationships among the new uncultivated microbes. Unlike Linneaus, Darwin or Haeckel, we organize geneologies based on genotype, not phenotype. This also gives us a picture of evolution. Note the organisms in blue — those we only know from environmental sequences or fingerprints. Also note how we can compare where they fall on the tree to known organisms. OK, let's talk more about our study of creatures from the Black Lagoon. IV. Evolutionary Implications of Uncultivated Eukaryotes Marine Sampling Sites So we've chosen three sites altogether in which to check for previously undiscovered microbes. Two of the sites are local: Bolinas Lagoon by Stinson Beach and the Berkeley Aquatic Park. We chose these two sites because the sediment had low oxygen levels. Freshwater Sampling Another site we've investigated is from a freshwater lake in Bloomington, Indiana. We chose a freshwater site in addition to the marine sites because they tend to have different types of protists than marine environments. So what have we found? Well, lots of types of protists. I've got plenty of trees, which I'll spare you, but below are some of the types of organisms we've found. I suppose if we were on their scale, they truly would be Creatures from the Black Lagoon. These are some examples of perhaps the most interesting eukaryotic microbes we've found, especially in the context of the evolution of eukaryotes.   Outside the "Crown" Many of the protists we've found are not closely related to any known group of protists. The "crown" refers to the most recent "big bang" in eukaryote evolution — where it seems a lot of groups of organisms evolved quickly, including our animal ancestors. Several of the new environmental sequences are deep in the tree, suggesting that they might have retained some characteristics of the ancestral eukaryotes. We think we've found about eight new kingdoms of eukaryotes in just three environments by just hunting for their DNA. Not bad for a teaspoon of stinky mud. These pictures are of Giardia (top left), Trichomonas (top middle), a slime mold (bottom left), Euglena (bottom middle) and several amitochondriate amoebae (top and bottom right). New Kingdoms of Eukaryotes Here's a comparison of trees with and without the new groups of eukaryotes. You'll note both the placement of the new branches (the mode or pattern of evolution) and the length of the branches (the rate or tempo of evolution). Models and Sampling Bias I think it's obvious that we need to reconsider our knowledge of extant eukaryotic microbes, those unseen and ungrown, living today all around us. Adding the few we've discovered into the tree, it really changes the picture of evolution. And in coming years, the picture will likely change a lot more. What's more, we can use our knowledge of the rRNA sequence as a tag to help identify them if we try to grow them — which would be worthwhile. Conclusions In understanding the history of life on Earth, it is first important to understand our own misconceptions. So, I've spoken about our own biases and misconceptions in evolution, life without oxygen, unicellular eukaryotes as opposed to plants and animals, and how to know organisms without first having to grow them, and lastly how to find new eukaryotic kingdoms in your own backyard... So, it's off to find new microbes — where should we look next? Boiling Hot Springs! We're thinking that eukaryotes might have adapted to other extreme environments other than those without oxygen. Maybe somewhere like this hot spring in Yellowstone National Park pictured at the left. Aside from being anoxic, the early Earth was also much warmer than today. The current idea is that Bacteria and Archaea arose during these hotter times. Why not eukaryotes?